N3 S2 chelating ligands optionally radiolabelled with Tc or Re, useful for diagnostic or therapeutic applications

ABSTRACT

Novel N 3  S 2  chelating ligands can be chelated to a radiodetectable element, Tc or Re, and are useful for radiolabeling peptides. A group of peptides especially useful are somatostatin derivatives which are valuable of the selective detection or treatment of tumors with somatostatin receptors.

BACKGROUND OF THE INVENTION

Radiopharmaceutical chelate systems have been used to couple withpeptides and proteins, for the purpose of diagnostic imaging. Many suchchelate systems have a central "N₂ S₂ ", "N₃ S₃ " moiety; a few have a"N₃ S₂ " moiety.

The N₃ S₂ containing system is preferred due to the combination of onecentral alkyl amino group and two other alkyl/amide nitrogens and twothiols which are responsible for the chelating properties, and whichform stable compounds with Group VIIb metals at reasonable temperatures(i.e., ≦60° C.,) and which also allow simple attachment to peptides andproteins under aqueous conditions.

SUMMARY OF THE INVENTION

The present invention relates to novel N₃ S₂ chelating ligands and aprocess for their production. These are useful for attachment topeptides and proteins, and thereafter chelated with appropriate radiometallic isotopes for treatment of appropriate receptor positive tumorsor as in vivo diagnostic imaging agents.

These chelates are "N₃ S₂ " type compounds of the following structure:##STR1## wherein R is hydrogen, loweralkyl of 1-4 carbon atoms, orloweralkyl carboxyl, wherein loweralkyl is 1-4 carbon atoms;

R¹ is hydrogen, a suitable protecting group such as p-loweralkyloxyl(1-4 carbon atoms) benzyl such as p-methoxylbenzyl; trityl; or R¹ and R¹are linked together to form a bond between the two "S" groups;

and R² is either a free amino or an isothiocyanato group, which can be##STR2## and n or m are independently an integer from 1-4.

These ligands are prepared by using either a cyclic approach, when R¹and R¹ are linked together to form the disulfide bond; or an open chainapproach when R¹ and R¹ are either hydrogen or the protecting group.

The cyclic approach reacts a tris(2-aminoethyl)amine with theappropriate dithio-dialdehyde in a suitable solvent such as loweralkanol at reflux. Thereafter, the cyclic amine is optionally reacted onthe secondary amine groups, to provide the desired R groups, andsubsequently with the appropriate N-hydroxy succinimide ester intriethylamine and dichloromethane to yield compounds in which R² is theamino group; and subsequently optionally reacted with thiophosgene toyield the corresponding isothiocyanato derivatives.

The open chain approach utilizes a diethylenetriamine reaction withfirst phthalic anhydride; second, p-nitrobenzyl bromide; and third, 6NHCl at reflux to yield the appropriateN'-(4-nitrobenzyl)-bis-(2'-phthalimidoethyl)amine, to which subsequentlythe pendent blocking groups are attached, followed by reaction of theamino group to isothiocyanato if desired.

The novel N₃ S₂ ligands can then be labelled with the appropriateradioisotope by reacting with the radioisotope in the form of a transferchelate such as a glucoheptonate complex (available commercially) andheating at 40°-80° C. for 1-4 hours. Alternatively, the N₃ S₂ ligandscan be coupled with the appropriate peptide or protein and thenradiolabelled using a similar procedure.

After labelling, the N₃ S₂ ligands are useful as radioimaging agents oras radio therapeutic agents for the treatment of appropriate tumors.When the ligands are to be first coupled with the peptide or protein,the isothiocyanate group at R² is first prepared for reaction with theamino group of the peptide or protein; and thereafter chelated with theappropriate radioisotope.

The preferred peptide or protein to be used includes natural andsynthetic somatostatin and analogues, atrial natriuretic factorpeptides, fibrin binding domain peptides, and monoclonal antibodies orfragments thereof, F(ab)₂, Fab, Fv regions; oxytocin; substance P;vasopression; as well as any peptidomimetics containing an amino group.

The preferred radioisotope is one of those of rhenium or technetium,preferably Re-186, Re-188 or Tc-m99.

Other radio isotopes can be used which can be any detectable element. Bydetectable element is meant any element, preferably a metal ion whichexhibits a property detectable in therapeutic or in vivo diagnostictechniques, e.g., a metal ion which emits a detectable radiation or ametal ion which is capable of influencing NMR relaxation properties, andwhich is capable of forming a chelate with the N₃ S₂ system.

Suitable detectable metal ions include, for example heavy elements orrare earth ions, e.g., paramagnetic ions, e.g., Gd³⁺, Fe³⁺, Mn²⁺ andCr²⁺, fluorescent metal ions, e.g., Eu³⁺, and radionuclides, e.g.,γ-emitting radionuclides, β-emitting radionuclides, α-emittingradionuclides, positron-emitting radionuclides, e.g., ⁶⁸ Ga.

The products of this invention are useful either as a diagnostic imagingagent, e.g., visualization of the particular (peptide) receptor positivetumors and metastases when complexed with a paramagnetic, a γ-emittingmetal ion or a positron-emitting radionuclide, or as a therapeuticradiopharmaceutical for the treatment in vivo of (peptide) receptorpositive tumors and metastases when complexed with a α- orβ-radionuclide, as indicated by standard tests.

The particular radioisotope chosen is relevant to the organ or system tobe radioimaged. For instance, in the last few years a high incidence ofsomatostatin receptors has been demonstrated in a variety of humantumors, e.g., pituitary tumors, central nervous system tumors, breasttumors, gastroenteropancreatic tumors and their metastases. Some of themare small or slow-growing tumors which are difficult to preciselylocalize by conventional diagnosis methods, but in vitro visualizationof somatostatin receptors has been performed through autoradiography oftumoral tissues using radioiodinated somatostatin analogues.

The products of this invention when used as imaging agents may beadministered parenterally, preferably intravenously, e.g., in the formof injectable solutions or suspensions, preferably in a singleinjection. The appropriate dosage will of course vary depending upon,for example, the precise chelating ligand and the type of detectableelement used, e.g., the radionuclide. A suitable dose to be injected isin the range to enable imaging by photoscanning procedures known in theart. It may advantageously be administered in a dose having aradioactivity of from 0.1 to 50 mCi, preferably 0.1 to 30 mCi, morepreferably 0.1 to 20 mCi. An indicated dosage range may be of from 1 to200 μg product labelled with 0.1 to 50 mCi, preferably 0.1 to 30 mCi,e.g., 3 to 15 mCi, γ-emitting radionuclide, depending on the γ-emittingradionuclide used.

The enrichment in the tumorigenic sites with the products may befollowed by the corresponding imaging techniques, e.g., using nuclearmedicine imaging instrumentation, for example a scanner, planar orrotating γ-camera, each preferably computer assisted; PET-scanner(Positron emission tomography) or MRI equipment.

These products can also be used for in vivo treatment of peptidereceptor positive tumors and metastases in a subject in need of such atreatment which comprises administering to said subject atherapeutically effective amount of the product.

Dosages employed in practicing the therapeutic method of the presentinvention will of course vary depending, e.g., on the particularcondition to be treated, for example the volume of the tumor, theparticular product employed, for example the half-life of the product inthe tumor, and the therapy desired. In general, the dose is calculatedon the basis of radioactivity distribution to each organ and on observedtarget uptake. For example, the product may be administered at a dailydosage range having a radioactivity of from 0.1 to 3 mCi/kg body weight,e.g., 1 to 3 mCi, preferably 1 to 1.5 mCi/kg body weight. An indicateddaily dosage range is of from 1 to 200 μg ligand labelled with 0.1 to 3mCi/kg body weight, e.g., 0.1 to 1.5/kg body weight α- or β-emittingradionuclide, conveniently administered in divided doses up to 4 times aday.

These products may be administered by any conventional route, inparticular parenterally, e.g., in the form of injectable solutions orsuspensions. They may also be administered advantageously by infusion,e.g., an infusion of 30 to 60 min. Depending on the site of the tumor,they may be administered as close as possible to the tumor site, e.g.,by means of a catheter. The mode of administration selected may dependon the dissociation rate of the product used and the excretion rate.

These products may be administered in free form or in pharmaceuticallyacceptable form, such as salts which may be prepared in conventionalmanner and exhibit the same order of activity as the free compounds.

The products for use in the method of the present invention maypreferably be prepared shortly before the administration to a subject,i.e., the radiolabelling with the desired detectable metal ion,particularly the desired α-, β- or γ- radionuclide, may be performedshortly before the administration.

They are then suitable for imaging or treating tumors such as pituitary,gastroenteropancreatic, central nervous system, breast, prostatic,ovarian or colonic tumors, small cell lung cancer, paragangliomas,neuroblastomas, pheochromocytomas, medullary thyroid carcinomas,myelomas, etc. and metastases thereof, as well as lymphomas.

According to a further aspect of the invention, there is provided:

i. a pharmaceutical composition comprising the radiolabelled product ofthe invention in free or in pharmaceutically acceptable salt form,together with one or more pharmaceutically acceptable carriers ordiluents therefor; or

ii. a pharmaceutical composition comprising a chelate-peptide productaccording to the invention in free or in pharmaceutically acceptablesalt form, together with one or more pharmaceutically acceptablecarriers or diluents therefor.

Such compositions may be manufactured in conventional manner.

A composition according to the invention may also be presented inseparate package with instructions for mixing the chelate-peptideproduct with the metal ion and for the administration of the resultingradiolabelled product. It may also be presented in twin-pack form, thatis, as a single package containing separate unit dosages of the ligandand the detectable metal ion with instructions for mixing them and foradministration of the product. A diluent or carrier may be present inthe unit dosage forms.

A preferred use of the N₃ S₂ ligands of this invention is then couplingto a novel synthetic somatostatin receptor-binding peptide. The bestsomatostatin receptor-binding peptide is a synthetic hexapeptide.##STR3## wherein Phe, Tyr, Trp, Lys, Thr, and Lys are the usual aminoacids. This and other suitable peptides are disclosed in Tel. Letters,Vol. 32, No. 36, pp. 4675-4678, 1991. This cyclic hexapeptide can belinked via the isothiocyanato moiety to any of the N₃ S₂ ligands in thisapplication, and then labelled with Tc-99. The chelated and/orradiolabelled product is not a part of this invention, but is disclosedand claimed in a separate application, Attorney docket Case 19312, filedsimultaneously with the instant application.

EXPERIMENTAL Materials and Methods

Unless otherwise specified, all reactions were carded out in oven-driedflasks at room temperature under an argon atmosphere with magneticstirring. After extraction, organic solvents were dried over MgSO₄,filtered, and removed under reduced pressure on a rotary evaporator.Reagent grade solvents, starting materials and deuterated solvents werepurchased from Aldrich Chemical Co. (Milwaukee, Wis.) and used withoutfurther purification.

¹ H NMR spectra were obtained on a Bruker Model AM 500, AM 400, and AM300. Samples were dissolved in CDCl₃, MeOD₄, or DMSO-d₆ and chemicalshifts were reported as δ values with the solvent or tetramethylsilaneresonance as the internal standard. The multiplicity is defined bys(singlet), d(doublet), t(triplet), q(quartet), and m(multiplet). Therelative peak heights of the resonances are reported as integersfollowing the multiplicity. ¹³ C NMR spectra were recorded on a BrukerAM-300 spectrometer at 75.5 MHz and the degree of substitution of eachcarbon atom was determined by complete decoupling and DEPT composed 135°pulsed sequence experiments. For ¹³ C the carbon and proton signals wereassigned by heterocorrelation experiments.

Infrared(IR) spectra (solution cells-CDCl₃ as solvent) were recorded ona Perkin Elmer 681 infrared spectrophotometer. Melting points weredetermined on a Thomas Hoover capillary melting point apparatus and areuncorrected. Mass spectra(MS) were recorded either in the CI(methanegas) or FAB mode using Finnigan 4500 single quadrupole mass spectrometerand were run by Oneida Research Services, Inc. (Whitesboro, N.Y.). Flashchromatography was performed essentially as described in the literature¹using Merck silica gel 60 (230-400 mesh) as stationary phase with theuse of the following solvents: methanol(M), methylene chloride(C),ammonium hydroxide(H).

Part 1 Cyclic Approaches to N₃ S₂ Chelates Synthesis of N₃ S₂-isothiocyanate (11)TPPBI3,3,13,13-Tetramethyl-1,2-dithia-5,8,11-triazacyclotridecan-8-yl-ethanamine(4)

A round bottom flask was charged with tris(2-aminoethyl)-amine (1) (989mg, 6.76 mmol), α,α'-dithiodiiso-butyraldehyde (2) (1,380 mg, 6.69 mmolprepared according to reference 2b) and ethanol (200 ml). The mixturewas stirred at room temperature for 1.5 hours and then refluxed for 3hours. After cooling the volatile materials were removed in vacuo togive the crude di-imine 3 as a glassy solid. ¹ H NMR analysis of thedi-imine 3 gave three singlets centered at 7.58 ppm (17:67:17)confirming the formation of an imine.

To the crude di-imine 3 in refluxing ethanol (200 mL) was added sodiumborohydride (1.346 g, 35.58 mmol) in two portions over 3.5 hours. Thereaction mixture was refluxed for a total of 17 hours and acetone (100ml) was added to destroy excess reagent. After cooling the solvent wasremoved in vacuo, water was added, and the product was extracted with 5%MeOH/CH₂ CL₂. The organic layer was dried (MgSO₄), filtered, andconcentrated in vacuo to give a yellow oil. Flashchromatography of thisoil using 82.5% C/15.0% M/2.5% H gave amine 4 as a light yellow viscousoil (1.268 g, 59.5% yield).

¹ H NMR (in CDCl₃): δ2.88 (t,j=5.9 Hz,2H,N-CH₂ CH₂ -NH₂), 2.72(s,4H,2N-CH₂ -C-S), 2.70 (m,4H,2N-CH₂ CH₂ -NH), 2.58 (m,4H,2N-CH₂ -CH₂-NH), 2.50 (t,j=5.9 Hz,2H,N-CH₂ CH₂ -NH₂), 1.73 (br s,4H,4NH), 1.33(s,12H,4CH₃ -C-S) ppm. ¹³ C NMR (in CDCl₃): 59.2 (t,2NH-CH₂ -C-S), 56.6(t,N-CH₂ CH₂ -NH₂), 54.5 (t,2N-CH₂ CH₂ -NH), 50.5 (s,2CH₃ -C-S), 47.4(t,2N-CH₂ CH₂ -NH), 39.6 (t,N-CH₂ CH₂ -NH₂), 27.4 (q,4CH₃ -C-S)ppm.MS(m/z,CI): 321(100, M⁺ +1). IR (CDCl₃): 3350, 2940, 2820, 1455, 1360,1120 cm⁻¹.

3,3,13,13-Tetramethyl-1,2-dithia-5,8,11-triazacyclotridecan-8-yl-(2'-N-phthaloyl)-ethanamine(5)

A round bottom flask was charged with amine 4 (111.2 mg, 0.347 mmol),N-carboethoxyphthalimide (108.3 mg, 0.494 mmol), and dichloromethane (10mL). The reaction mixture was stirred at room temperature for 1 hour andthe solvent was removed in vacuo. Flash chromatography of the residueusing 90% C/9.5% M/0.5% H gave the phthalate 5 as a yellowish oil (119.2mg, 76.3%).

¹ H NMR (in CDCl₃): δ7.86 (m,2H,H₂ &H₅ -Ar), 7.71 (m,2H,H₃ &H₄ -Ar),3.84 (t,j=6.4 Hz,2H,N-CH₂ CH₂ -NPhth), 2.81 (t,j=6.4 Hz,2H,N-CH₂ CH₂-NPhth), 2.69 (m,4H,2N-CH₂ CH₂ -NH), 2.66 (m,4H,2N-CH₂ CH₂ -NH), 2.59(s,4H,2N-CH₂ -C-S), 1.87 (br m,2H,2NH), 1.23 (s,12H,4CH₃ -C-S) ppm. ¹³ CNMR (in CDCl₃): 168.1 (s,2CO), 133.7 (d,C₃ &C₄ -Ar), 131.9 (s,C₁ &C₆-Ar), 123.3 (d,C₂ &C₅ -Ar), 58.7 (t,₂ NH-CH₂ -C-S), 53.9 (t,2N-CH₂ CH₂-NH), 52.8 (t,N-CH₂ CH₂ -NPhTh), 50.2 (s,2CH₃ -C-S), 47.6 (t,2N-CH₂ CH₂-NH), 35.8 (t,N-CH₂ CH₂ -NPhTh), 27.3 (q,4CH₃ -C-S) ppm. MS (m/z,CI):451(100, M⁺ +1). IR (CDCl₃): 3400, 2950, 2810, 1770, 1705, 1465, 1395cm⁻¹.

3,3,5,11,13,13-Hexamethyl-1,2-dithia-5,8,11-triazacyclotridecan-8-yl-ethanamine(7)

A round bottom flask was charged with phthalate 5 (775.7 mg, 1.72 mmol),formic acid (10 mL, 265 mmol), and formaldehyde (37% wt in water, 15 mL,200 mmol). The reaction mixture was refluxed for 20 hours and thenallowed to cool and the solvent was removed in vacuo. A solution of 10%KOH was added to the solid residue and the compound extracted with 5%MeOH/CH₂ Cl₂. The organic solvent was dried (MgSO₄), filtered, andconcentrated in vacuo to give crude 6 as a viscous oil.

To a round bottom flask was added crude 6, hydrazine monohydrate (1 mL,20.6 mmol), and ethanol (40 mL). The reaction mixture was refluxed for20 hours and then allowed to cool. The solvent was removed in vacuo,water added, and the residue extracted with 5% MeOH/CH₂ CL₂. The organicsolvent was dried (MgSO₄), filtered, and concentrated in vacuo to give ayellowish viscous oil. Flash chromatography of the crude product using80% C/19.5 % M/1.0% H gave dimethyltetramine 7 as a light yellow oil(375 mg, 62.4% overall yield from 4).

¹ H NMR (in CDCl₃): δ2.78 (t,j=5.9 Hz,2H,N-CH₂ CH₂ -NH₂), 2.72 (t,j=5.5Hz,4H,2N-CH₂ CH₂ -NCH₃), 2.63 (s,4H,2N-CH₂ -C-S), 2.60 (t,j=5.5Hz,4H,2N-CH₂ CH₂ -NCH₃), 2.42 (t,j=5.9 Hz,2H,N-CH₂ CH₂ -NH₂), 2.36(s,6H,2NCH₃), 1.77 (br s,2H,NH₂), 1.28 (s,12H,4CH₃ -C-S) ppm. ¹³ C NMR(in CDCl₃): 67.2 (t,2N-CH₂ -C-S), 57.9 (t,2N-CH₂ CH₂ -NCH₃), 55.6(t,N-CH₂ CH₂ -NH₂), 52.1 (t,2N-CH₂ CH₂ -NCH₃), 51.3 (s,2CH₃ -C-S) 45.1(q,2NCH₃), 39.5 (t,N-CH₂ CH₂ -NH₂), 27.4 (q,4CH₃ -C-S) ppm. MS (m/z,CI):349(100,M⁺ +1). IR (CDCl₃): 2960, 2800, 1455, 1355, 1310, 1100 cm⁻¹.

4-Amino-N-[2-(3,3,5,11,13,13-hexamethyl-1,2-dithia-5,8,11-triazacyclo-tridecan-8-yl)ethyl]benzamide(10)

A round bottom flask was charged with the dimethyl-tetramine 7 (514.9mg, 1.48 mmol), tBoc-p-aminobenzoyl N-hydroxysuccinimide ester (8)(493.8 mg, 1.48 mmol, prepared according to reference 3), triethylamine(0.21 mL, 1.51 mmol), and dichloromethane (40 mL). The reaction mixturewas stirred at room temperature for 12 hours and the solvent was removedin vacuo to give crude 9.

To a round bottom flask was added crude 9, dichloromethane (10 mL), andtrifluoroacetic acid (10 mL) and the resulting mixture was stirred atroom temperature for 1 hour. The solvent was removed in vacuo andpurification by flash chromatography of the residue using 94.5% C/5.0%M/0.5% H gave the aniline 10 as a yellowish white solid (345.8 mg, 50.8%overall yield from dimethyltetramine 7).

¹ H NMR (in CDCl₃): δ7.65 (d,j=8.6 Hz,2H,H₂ &H₆ -Ar), 6.87 (brs,1H,NH-CO), 6.67 (d,j=8.6 Hz,2H,H₃ &H₅ -Ar), 3.95 (s,2H,NH₂), 3.49(t,j=5.4 Hz,2H,N-CH₂ CH₂ -NCO), 2.74 (m,4H,2N-CH₂ CH₂ -NCH₃), 2.65(m,4H,2N-CH₂ CH₂ -NCH₃), 2.61 (s,4H,2N-CH₂ -C-S), 2.53 (t,j=5.4Hz,2H,N-CH₂ CH₂ -NCO), 2.30 (s,6H,2NCH₃), 1.29 (s,12H,4CH₃ -C-S) ppm. ¹³C NMR (in CDCl₃): 167.1 (s,NHCO), 149.6 (s,C₁ -Ar), 128.6 (d,C₃ &C₅-Ar), 123.9 (s,C₄ -Ar), 113.9 (d,C₂ &C₆ -Ar), 67.2 (t,N-CH₂ -C-S), 57.6(t,2N-CH₂ CH₂ -NCH₃), 53.7 (t,N-CH₂ CH₂ -NHCO), 53.4 (s,2CH₃ -C-S), 51.4(t,2N-CH₂ CH₂ -NCH₃) 45.0 (q,2NCH₃), 37.0 (t,N-CH₂ CH₂ -NHCO), 27.2(q,4CH₃ -C-S) ppm. MS(m/z,FAB): 468.2(100, M⁺ +1). IR (CDCl₃): 3405,2960, 2800, 1620, 1495, 1280, 1100, 835 cm⁻¹.

4-Isothiocyanato-N-[2-(3,3,5,11,13,13-hexamethyl-1,2-dithia-5,8,11-triazacyclotridecan-8-yl)ethyl]benzamide(11) [TPPBI]

To a round bottom flask with the aniline 10 (53.3 mg, 0.114 mmol) anddichloromethane (5 mL) was added 0.2007M solution of thiophosgene (0.60mL, 0.120 mmol) in dichloromethane. The heterogeneous reaction mixturewas stirred at room temperature for 1 hour and the solvent was removedin vacuo to give the isothiocyanate 11 as a reddish solid (67.8 mg,116%).

¹ H NMR (in DMSO-d₆): δ8.88 (br s,1H,NH-CO), 7.99 (d,j=8.4 Hz,2H,H₂ &H₆-Ar), 7.54 (d,j=8.4 Hz,2H,H₃ &H₅ -Ar), 3.49 (m,2H,N-CH₂ CH₂ -NCO), 3.40(m,8H,2N-CH₂ CH₂ -NCH₃), 3.01 (s,4H,2N-CH₂ -C-S), 2.89 (s,6H,2NCH₃),2.71 (m,2H,N-CH₂ CH₂ -NCO), 1.45 (s,12H,4CH₃ -C-S) ppm. MS (m/z,CI):510(100, M⁺ +1). IR (CDCl₃): 3400, 2960, 2100, 1640, 1600, 1545, 1500,1470, 1300 cm⁻¹.

Preparation of Somatoscan(Fmoc)TPPBI (13)

In a 5 mL Reacti-Vial (Pierce) a solution of SomatoscanFmoc (12)[cyclo(Trp-Lys(Fmoc)-Val-Lys-NMe-Phe-Tyr)](1.97 mg; 1.515 umol), TPPBI(11) (8.4 mg, 16.495 umol), and DMF (400 μL) was stirred whilebicarbonate/phosphate buffer (0.2M, pH 8.2, 100 μL, freshly prepared)was added. The heterogeneous solution was monitored by HPLC and stirredfor 4 hours at room temperature. The solvents were removed in vacuo andthe residue was partitioned with 1N HCl (300 μL) and MeOH (100 μL).Purification of this solution by HPLC (Hamilton PRP-1 12-20 μmpreparative column 250×21.5 mm) using a 30% to 100% gradient ofacetonitrile: water (containing 0.1% TFA) over 40 minutes (flow rate of12 mL/min) afforded pure Somatoscan(Fmoc)TPPBI (13) (R_(t) =20.64 min).

MS (Electrospray, Hypermass)799.4(z=2), Calc. Compound Mass=1596.8,Meas. Compound Mass=1597.8.

Part 2 Acyclic Approaches to N₃ S₂ Chelates Synthesis of AcyclicDimercaptoanisidine Arylisothiocyanates Synthesis of open chain N₃ S₂-aryl isothiocyanate (21) Bis(2'-phthalimidoethyl)amine, (15)

This was prepared according to reference 4.

Phthalic anhydride (32 g; 0.22 mol) was dissolved in 333 mL of hotchloroform and the mixture was filtered to eliminate phthalic acid*. ADiethylenetriamine 14 (7.97 g; 0.077 mol) solution in chloroform (64 mL)was slowly added (over a period of 50 minutes) to the phthalic anhydridemixture maintained at a temperature of 50° C. Temperature was raised to110° C. after the addition was over. The reaction mixture was thenstirred for 48 hours and slowly concentrated. The concentrate solutionwas then treated with activated charcoal. 31.8 g of a yellow solid wasrecovered after evaporation of the solvent under reduced pressure. Thesolid was triturated successively with ether, ethanol and then dissolvedin methylene chloride. The methylene chloride solution was washed with10% sodium carbonate (3×500 mL), water and saturated sodium chloridesolution. The organic phase was dried with magnesium sulfate, filteredand evaporated to dryness under reduced pressure. A pale yellow solid(14.47 g; 52%) was obtained. A portion (4.45 g) of that product waspurified by flash chromatography (silica gel) using a mixture ofmethylene chloride, ethyl acetate and triethylamine as elution system(79/20/1). The purification give 2.798 g of bis(phthalimidoethyl)amine(15). * 6.96 g of phthalic acid was recovered.

¹ H NMR (in CDCl₃): δ7.70 (m,8H,H-Ar(phth)), 3.77 (t,J=6 Hz,4H,--NH(-CH₂ -CH₂ -NPhth)₂), 2.95 (t,J=6 Hz,4H,-NH(-CH₂ -CH₂ -NPhth)₂),1.41 (broad,1H,-NH(-CH₂ -CH₂ -NPhth)₂) ppm. IR (in CDCl₃ /NaCl): 3460(N-H,w, sec amine), 2940-2820 (C-H), 1770-1710 (C=O, Phth), 1465, 1425,1390, 1360, 1185, 1035 cm⁻¹ MS (EI; m/z): 363(0.4,M⁺), 364(4,M⁺ +1),216(3,M⁺ -Phth ), 204(18), 203(100,M⁺ -(Phth-CH₂ •)), 174(57,Phth-CH₂-CH₂ ⁺), 160(5), 147(6), 130(12) and 56(6).

N'-(4-Nitrobenzyl) bis(2'-phthalimidoethyl)amine (16)

(See Ref. 4) In a 250 mL round bottom flask potassium hydroxyde (1.6 g;28 mmol) was dissolved in hot ethanol (100 mL). To that ethanolicsolution Bis(2'-phthalimidoethyl)amine (15) (10.02 g; 28 mmol) wasadded. The solution was magnetically stirred and refluxed for 21/2 hoursbefore p-nitrobenzyl bromide (5.95 g; 28 mmol; 1 eq) was added. Thereaction mixture was heated at reflux for 16 additional hours thenfiltered hot. The solid obtained previously was washed with absoluteethanol and dried under vacuum to yield 7.441 g (54%) of a white solid(p-nitrobenzyl bisphthalimide). The flitrate was evaporated underreduced pressure to give 8.19 g of a yellow solid. That residue waspurified by flash chromatography (silica gel: 400 g) using methylenechloride-methanol (98/2) system as eluent. The purification bychromatography produced 3.13 g (23%) of the desired product. Thealkylation reaction yielded 10.571 g ofN'-(4-nitrobenzyl)bis(2'-phthalimidoethyl)amine. (16)

¹ H NMR (in CDCl₃): a 7.70 (m,10H,H-Ar(Phth)+o(H)-Ar-NO₂), 7.20 (d, J=9Hz,2H, m(H)-Ar-NO₂), 3.75 (t, J=6 Hz, 4H,-NH (-CH₂ -CH₂ -NPhth)₂), 3.71(s, 2H,-N-CH₂ -Ar-NO₂) and 2.80 (t, J=6 Hz,4H,-NH(-CH₂ -CH₂ -NPhth)2)ppm. MS (El; m/z): 498(1,M⁺), 499(0.6,M⁺ +1), 362(1,M⁺ -·CH₂ Ar-NO2),339 (32, M⁺ +1-(Phth-CH₂ •)), 338 (100, M⁺ -(Phth-CH₂ ·)), 324 (2, M⁺-(Phth-CH₂ -CH₂ •)), 174(58,Phth-CH₂ -CH₂ ⁺), 173(42), 165(6), 163(8),161(6), 160(43), 149(12), 136(24), 130(12), 106(21), 105(12), 104(17),90(22), 89(18), 78(23), 77(21) and 76(12).

Hydrolysis of N'-(4-nitrobenzyl)bis(2'-phthalimidoethyl)amine

In a 250 mL round bottom flask, provided with a condenser,N'-(4-nitrobenzyl) bis(2'-phthalimidoethyl)amine (16) (2.80 g; 5.62mmol) and 6N hydrochloric acid (150 mL) were introduced. The reactionmixture was stirred and refluxed for 23 hours. The solution was cooledwith an ice bath and filtered. The filtrate was washed with ether (3×100mL) and dried by vacuum to give a yellow foam-like material (2.17 g).The residue was dissolved in water (10 mL) and the pH of that solutionwas brought basic with 1N sodium hydroxide (25 mL). Then the mixture wasextracted with methylene chloride (3×75 mL). The organic extracts werecombined, dried with magnesium sulfate, filtered and evaporated todryness to yield 1.347 g of N'-(4-nitrobenzyl)bis(2'-aminoethyl)amine(17) as a light orange oil (which turn dark red with time).

Note: The p-nitrobenzyltriamine (17) is stored for short term away fromlight and in an inert atmosphere of argon. For long term storage it isbetter to keep that compound as the hydrochlorate form.

¹ H-NMR (in CDCl₃): δ8.13 (d,J=9 Hz,2H,o(H)-Ar-NO₂), 7.46 (d, J=9 Hz,2H,m(H)-Ar-NO₂), 3.65 (s,2H,-N-CH₂ -Ar-NO₂), 2.74 (t,J=6 Hz, 4H, -N(-CH₂-CH₂ -NH₂)₂), 2.50 (t,J=6 Hz,4H,-N(-CH₂ -CH₂ -NH₂)₂) and 1.43 (broads,4H, -N(-CH₂ -CH₂ -NH₂)₂)ppm. IR (film): 3370-3290 (N-H,-NH₂),2940-2800(C-H), 1605 (C=C,Ar), 1510 (N=O,Ar), 1450, 1340 (N=O,Ar), 1105,1010, 850 (C-N,Ar-NO₂) and 730 cm⁻¹.

N'-4-Aminobenzyl-diethylenetriamine (18)

This was prepared according to reference 4. ¹ H NMR (in CDCl₃): δ7.08(d,j=8.3 Hz,2H,H₃ &H₅ -Ar), 6.64 (d,j=8.3 Hz,2H,H₂ &H₆ -Ar), 3.62 (brs,2H,Ar-NH₂), 3.48 (s,2H,N-CH₂ -Ar), 2.74 (t,j=6.0 Hz,4H,2N-CH₂ CH₂-NH₂), 2.50 (t,j=6.0 Hz,4H,2N-CH₂ CH₂ -NH₂), 1.52 (br s,4H,2NH₂) ppm.

N,N"-Bis[2-((4-methoxybenzyl)thio)-2-methyl-propionyl]-N'-(4-aminobenzyl)-diethylenetriamine(20)

To a solution of the aniline 18 (230 mg, 1.10 mmol, freshly prepared) inethanol (20 mL) was added a solution of2-[(p-methoxy-benzyl)thio]-2-methylpropionic acid chloride 19 (1.33 g,5.10 mmol, prepared according to reference 5) in dichloromethane (10 mL)over 15 minutes. The resulting solution was stirred for 48 hours and thesolvent was removed in vacuo, 1N NaOH was added, and the product wasextracted with CH₂ Cl₂. The organic layer was washed with water, dried(MgSO₄), filtered, and concentrated in vacuo to give a red oil. Flashchromatography of this oil using 5% MeOH/CH₂ Cl₂ gave the aniline 20 asa yellow oil (126. mg, 17.5% yield).

¹ H NMR (in CDCl₃): δ7.15 (d,j=8.6 Hz,4H,2H₃ H₅ -Ar-OCH₃), 7.07 (t,j=8.2Hz,2H,H₃ &H₅ -Ar-NH₂), 7.07 (m,2H,2NHCO), 6.78 (d,j=8.6 Hz,4H,2H₂ &H₆-Ar-OCH₃), 6.58 (d,j=8.2 Hz,2H,H₂ &H₆ -Ar-NH₂), 3.74 (s,6H,2CH₃₀), 3.72(br s,2H,NH₂), 3.65 (s,4H,2S-CH₂ -Ar), 3.49 (s,2H,N-CH₂ -Ar), 3.24(q,j=5.9 Hz,4H,2N-CH₂ CH₂ -NHCO), 2.55 (t,j=6.2 Hz,4H,2N-CH₂ CH₂ -NHCO),1.50 (s, 12H,4CH₃ -C-S) ppm. ¹³ C NMR (in CDCl₃): δ174.6 (s,2NCO), 158.9(s,2C₁ -Ar-OCH₃), 146.0 (s,C₁ -Ar-NH₂), 130.2 (d,C₃ &C₅ -Ar-NH₂ +2C₃ &C₅-Ar-OCH₃), 129.6 (s,2C₁ &C₄ -Ar-OCH₃), 129.1 (s,C₄ -Ar-NH₂), 115.4 (d,C₂&C₆ -Ar-NH₂), 114.3 (d,2C₂ &C6-Ar-OCH₃), 58.3 (t,N-CH₂ -Ar), 55.5(q,2CH₃ O), 53.0 (t,2N-CH₂ CH₂ -NHCO), 50.3 (s,2S-C-CH₃), 37.8 (t,2N-CH₂CH₂ -NHCO), 34.4 (t,2S-CH₂ -Ar), 27.1 (q,4CH₃ -C-S) ppm. MS (m/z,FAB):653.5(100, MH⁺). IR (CDCl₃): 3380, 3000, 2930, 2835, 1665, 1510, 1245,1195, 1175, 1035 cm⁻¹.

N,N"-Bis[2-((4-methoxybenzyl)thio)-2-methyl-propionyl]-N'-(4-isothiocyanatobenzyl)-diethylenetriamine (21)

To a round bottom flask with the aniline 20 (55.7 mg, 0.0853 mmol) anddichloromethane (5 mL) was added 0.2011M solution of thiophosgene (0.42mL, 0.0845 mmol) in dichloromethane. The heterogeneous reaction mixturewas stirred at room temperature for 1 hour and the solvent was removedin vacuo to give the isothiocyanate 21 as a brown solid (68.9 mg, 116%).

¹ H NMR (in CDCl₃): δ7.72 (m,2H,2NHCO), 7.65 (d,j=8.3 Hz,2H,H₂ &H₆-Ar-NCS), 7.25 (t,j=8.3 Hz,2H,H₃ &H₅ -Ar-NCS), 7.17 (d,j=8.5 Hz,4H,2H₃H₅ -Ar-OCH₃), 6.80 (d,j=8.5 Hz,4H,2H₂ &H₆ -Ar-OCH₃), 4.14 (s,2H,N-CH₂-Ar), 3.77 (s,6H,2CH₃ O), 3.70 (s,4H,2S-CH₂ -Ar), 3.58 (m,4H,2N-CH₂ CH₂-NHCO),3.01 (m,4H,2N-CH₂ CH₂ -NHCO), 1.53 (s,12H,4CH₃ -C-S) ppm. IR(CDCl₃): 3300, 2930, 2080, 1655, 1605, 1510, 1245, 1170, 1030 cm⁻¹.

Part 3 Synthesis of Acyclic Dimercaptoanisidine Alkyl Isothio-cyanatesSynthesis of Open Chain N₃ S₂ -alkylanisidine 26N,N-Bis(2-aminoethyl)-N'-tert-butyl-oxycarbonyl-1,2-ethanediamine (22)

A solution of tris(2-aminoethyl)amine (1) (19.5 g, 133.6 mmol) in CH₂Cl₂ (300 mL) was cooled to -78° C. in a dry ice-acetone bath whiledi-tert-butyl dicarbonate (14.6 g, 66.9 mmol) in CH₂ Cl₂ (100 mL) wasadded slowly over 30 minutes. The reaction mixture was slowly allowed towarm up to room temperature and stirred for 18 hours. 1N NaOH was addedand the organic phase was dried (MgSO₄), filtered, and concentrated invacuo to give the amine 22 as a light yellow oil (9.07 g, 55%).

¹ H NMR (in CDCl₃): a 5.62 (br m,1H,NH-CO), 3.18 (m,2H,N-CH₂ CH₂ -NCO),2.98 (m,4H,2N-CH₂ CH₂ -NH₂), 2.80 (m,4H,2N-CH₂ CH₂ -NH₂), 2.57(m,2H,N-CH₂ CH₂ -NHCO), 2.57 (m,4H,2NH₂), 1.44 (s,9H,3CH₃ -C-O) ppm. MS(m/z,CI): 247 (100, MH⁺). IR (CDCl₃): 3280, 2965, 2815, 1695, 1500,1165, 905, 730 cm⁻¹.

N,N"-Bis[2-((4-methoxybenzyl)thio)-2-methyl-propionyl]-N'-[2-(N-tert-butoxycarbonyl)aminoethyl]-diethylenetriamine(23)

A solution of the ^(t) Boc derivative 22 (555.3 mg, 2.25 mmol) in CH₂Cl₂ (25 mL) was cooled to 0° C. while2-[(p-methoxy-benzyl)thio]-2-methylpropionic acid chloride 19 (1.54 g,6.85 mmol, prepared according to reference 5) in dichloromethane (10 mL)was added over 5 minutes. The resulting solution was allowed to warm toroom temperature and stirred for 12 hours. The solvent was removed invacuo, 1N NaOH was added, and the product was extracted with CH₂ Cl₂.The organic layer was washed with water, dried (MgSO₄), filtered, andconcentrated in vacuo to give a yellow oil. Flash chromatography of thisoil using 5% MeOH/CH₂ Cl₂ gave the ^(t) Boc derivative 23 as a yellowoil (938 mg, 60.2% yield).

¹ H NMR (in CDCl₃): δ7.16 (d,j=8.5 Hz,4H,2H₃ H₅ -Ar-OCH₃), 7.07 (t,j=5.2Hz,2H,2NH-CO), 6.81 (d,j=8.5 Hz,4H,2H₂ &H₆ -Ar-OCH₃), 4.98 (brs,1H,NH-CO), 3.77 (s,6H,2CH₃ O), 3.63 (s,4H,2S-CH₂ -Ar), 3.17(m,6H,3N-CH₂ CH₂ -NHCO), 2.56 (m,6H,3N-CH₂ CH₂ -NHCO), 1.53 (s,12H,4CH₃-C-S), 1.42 (s,9H,3CH₃ -C-O) ppm. MS (m/z,CI): 691 (9, MH⁺). IR (CDCl₃):3380, 2970, 2930, 2830, 1700, 1650, 1500, 1245, 1170, 1030, 830 cm⁻¹.

N,N"-Bis[2-((4-methoxybenzyl)thio)-2-methyl-propionyl]-N'-(2-aminoethyl)-diethylenetriamine(24)

A solution of 23 (858.3 mg, 1.24 mmol) in 50% TFA in CH₂ Cl₂ (30 mL) wasstirred at room temperature for 1 hour. The solvent was removed invacuo, 1N NaOH was added, and the product was extracted with CH₂ Cl₂.The organic layer was washed with water, dried (MgSO₄), filtered, andconcentrated in vacuo to give a yellow oil. Flash chromatography of thisoil using 90.5% C/9.5% M/0.5% H gave the amine 24 as a yellow oil (734mg, 95.1% yield).

¹ H NMR (in CDCl₃): δ7.25 (m,2H,2NH-CO), 7.16 (d,j=8.5 Hz,4H,2H₃ H₅-Ar-OCH₃), 6.81 (d,j=8.5 Hz,4H,2H₂ &H₆ -Ar-OCH₃), 3.77 (s,6H,2CH₃ O),3.69 (s,4H,2S-CH₂ -Ar), 3.23 (q,j=6.1 Hz,4H,2N-CH₂ CH₂ -NHCO), 2.72(t,j=5.9 Hz,2H,N-CH₂ CH₂ -NH₂), 2.54 (m,6H,N-CH₂ CH₂ -NH₂ +2N-CH₂ CH₂-NH-CO), 1.60 (br s,2H,NH₂), 1.52 (s,12H,4CH₃ -C-S) ppm. ¹³ C NMR (inCDCl₃): δ174.3 (s,2NH-CO), 158.3 (s,2C₁ -Ar-OCH₃), 129.6 (d,2C₂ &C₆-Ar-OCH₃), 128.9 (s,2C₄ -Ar-OCH₃), 113.6 (d,2C₃ &C₅ -Ar-OCH₃), 54.8(q,2CH₃ O), 54.2 (t,N-CH₂ CH₂ -NH₂), 53.6 (t,2N-CH₂ CH₂ -NHCO), 49.4(s,2S-C-CH₃), 38.5 (t,2N-CH₂ CH₂ -NHCO), 37.6 (t,N-CH₂ CH₂ -NH₂), 33.6(t,2S-CH₂ -Ar), 26.4 (q,4CH₃ -C-S) ppm. MS (m/z,CI): 591(31, MH⁺). IR(CDCl₃): 3770, 2930, 2830, 1655, 1510, 1245, 1170, 1030, 830 cm⁻¹.

N,N"-Bis[2-((4-methhoxybenzyl)thio)-2-methyl-propionyl]-N'-(2-isothiocyanoethyl)-diethylenetdamine(25)

To a round bottom flask containing the aniline 24 (28.8 mg, 0.0487 mmol)and dichloromethane (10 mL) was added 0.2211M solution of thiophosgene(0.22 mL, 0.0509 mmol) in dichloromethane. The heterogeneous reactionmixture was stirred at room temperature for 1 hour and the solvent wasremoved in vacuo. Flash chromatography of this crude product using 100%EtOAc gave the isothiocyanate 25 as a clear oil (18.9 mg, 61.3% yield).

¹ H NMR (in CDCl₃): δ7.17 (d,j=8.5 Hz,4H,2H₃ H₅ -Ar-OCH₃), 7.07(m,2H,2NH-CO), 6.82 (d,j=8.5 Hz,4H,2H₂ &H₆ -Ar-OCH₃), 3.78 (s,6H, 2CH₃O), 3.70 (s,4H,2S-CH₂ -Ar), 3.49 (t,j=5.9 Hz,2H,N-CH₂ CH₂ -NCS), 3.21(q,j=6.2 Hz,4H,2N-CH₂ CH₂ -NHCO), 2.78 (t,j=5.9 Hz,2H,N-CH₂ CH₂ -NCS),2.58 (t,j=6.4 Hz,4H,2N-CH₂ CH₂ -NHCO), 1.54 (s,12H,4CH₃ -C-S) ppm. MS(m/z,CI): 633(25, MH⁺). IR (CDCl₃): 3370, 2950, 2920, 2850, 2100, 1720,1655, 1610, 1510, 1245, 1170, 1030, 830 cm⁻¹.

N,N"-Bis[2-((4-methoxybenzyl)thio)-2-methyl-propionyl]-N'-(2-[[[4-methoxyphenyl)amino]thioxomethyl]aminoethyl]-diethylenetriamine(26)

A solution of the isothiocyanate 25 (18.9 mg, 0.02986 mmol),triethylamine (7.26 mg, 0.07175 mmol), anisidine (6.9 mg, 0.05602 mmol),and CHCl₃ (15 mL) was refluxed for 3 hours and the solvent was removedin vacuo. Flash chromatography of this crude product using 100% EtOAcgave the anisidine derivative 26 as a clear oil (19.8 mg, 87.6% yield).

¹ H NMR (in CDCl₃): δ8.17 (brs,1H,NH-CS-NH-Ar), 7.28 (d,j=8.8 Hz,2H,H₂&H₆ -Ar-N),7.15 (d,j=8.6 Hz,4H,2H₃ &H₅ -AroOCH₃), 7.06(brs,1H,NH-CS-NH-Ar), 7.06 (t,j=5.7 Hz,2H,2NH-CO), 6.90 (d,j=8.9Hz,2H,H₃ &H₅ -Ar-N), 6.82 (d,j=8.6 Hz,4H,2H₂ &H₆ -Ar-OCH₃), 3.79(s,3H,CH₃ O-Ar-N), 3.77 (s,6H,2CH₃ O-Ar-CH₂), 3.67 (s,4H,2S-CH₂ -Ar),3.61 (q,j=5.3 Hz,2H,N-CH₂ CH₂ -NHCS), 3.14 (q,j=6.2 Hz,4H,2N-CH₂ CH₂-NHCO), 2.69 (t,j=5.6 Hz,2H,N-CH₂ CH₂ -NHCS), 2.52 (t,j=6.3 Hz,4H 2N-CH₂CH₂ -NH-CO), 1.51 (br s,12H,4CH₃ -C-S)ppm. ¹³ C NMR (in CDCl₃): δ181.9(s,N-CS-N), 175.2 (s,2NH-CO), 158.8 (s,3C₁ -Ar-OCH₃), 130.0 (s,C₁-Ar-N), 130.0 (d,2C₂ &C₆ -Ar-OCH₃), 129.2 (s,2C₄ -Ar-OCH₃), 127.0 (d,C₂&C₆ -Ar-N), 114.5 (d,C₃ &C₅ -Ar-N), 114.2 (d,2C₃ &C₅ -Ar-OCH₃), 55.4(q,3CH₃ O), 54.4 (t,2N-CH₂ CH₂ -NHCO), 52.9 (t,N-CH₂ CH₂ -NHCS), 50.1(s,2S-C-CH₃), 42.8 (t.N-CH₂ CH₂ -NHCS), 38.3 (t,2N-CH₂ CH₂ -NHCO), 34.2(t,2S-CH₂ -Ar), 28.3 (q,4CH₃ -C-S) ppm. MS (m/z,FAB): 756(4.2, M⁺). IR(CDCl₃): 3320, 2960, 1720, 1655, 1510, 1290, 1245, 1030 cm⁻¹.

Part 4 Labeling of an N₃ S₂ Chelate Synthesis of AcyclicDimercapto-N,N,N-tris(2-aminoethyl)amine Preparation ofN,N-dimethyl-2-(3,3,5,11,13,13-hexamethyl-1,2-dithia-5,8,11-triaza-cyclotridecan-8-yl)ethylamine(27)⁶

To 2-(3,3,13,13-tetramethyl-1,2-dithia-5,8,11-triaza-cyclotridecan-8-yl)ethylamine 4(202.6 mg, 0.63 mmol), in a 25 mL round bottom flask, concentratedformic acid (4 mL) and 37% formaldehyde solution (3.7 mL) was added. Themixture was heated under reflux and stirred for 25 hours. The solutionwas then cooled to room temperature and extracted three times with ether(50 mL). The aqueous phase was rendered basic (˜10-11) by adding 27%ammonium hydroxide and extracted with methylene chloride (4×50 mL). Theorganic phase was washed successively with water (50 mL) and saturatedsodium chloride solution (2×50 mL), dried with anhydrous magnesiumsulfate, and filtered. The solvent was removed under vacuum to give 176mg (74%) of tetramethylated tetraamine disulfide 27 as a yellow oil. Thecrude product was purified by flash chromatography (silica gel) using amixture of methylene chloride, methanol, and ammonium hydroxide(84.5/15/0.5) as eluent. PureN,N-dimethyl-2-(3,3,5,11,13,13-hexamethyl-1,2-dithia-5,8,11-triaza-cyclotridecan-8-yl)ethylamine27 (108 mg) was recovered from the purification process.

N,N-dimethyl-2-(3,3,5,11,13,13-hexamethyl-1,2-dithia-5,8,11-triaza-cyclotridecan-8-yl)ethylamine27

¹ H NMR (300 MHz,in CDCl₃): δ2.71 (t,J=6 Hz,4H,-N-CH₂ -CH₂ -N(CH₃)-),2.64 (s,4H,-N-CH₂ -C(CH₃)2-S-), 2.62 (t,J=6 Hz,4H,-N-CH₂ -CH₂ -N(CH₃)-),2.52 (m,2H,-CH₂ -CH₂ -N(CH₃)₂), 2.46 (m,2H,-CH₂ -CH₂ -N(CH₃ (₂), 2.37(s,6H,-CH₂ -N(CH₃)-CH₂ -), 2.25 (s,6H,-CH₂ -CH₂ -N(CH₃)₂) and 1.28(s,12H,-S-C(CH₃)₂ -) ppm. ¹³ C NMR (75 MHz, in CDCl₃): δ74.8, 67.7,57.6, 57.2, 53.9, 51.7, 51.3, 45.9, 45.1 and 26.9 ppm. MS (El; m/z):376(4,M⁺), 377(2,M⁺ +1), 378(0.6,M⁺ +2), 318(1,M⁺ -((CH₃)₂ N-CH₂ •)),262 (3), 255 (1), 246(1), 187 (7), 156(9), 144(8), 133(9), 130(16), 113(17), 101(27), 99(21), 98(12), 72(32), 71(28), 70(42), 58(100), 56(13),55(13), 43(31), and 42(38).

Reduction ofN,N-dimethyl-2-(3,3,5,11,13,13-hexamethyl-1,2-dithia-5,8,11-triaza-cyclotridecan-8-yl)ethylamine (27)⁶

In a 50 mL three-necked flask equipped with a gas inlet device, amagnetic stirring bar, a stopper and a dry ice condenser 18.9 mg (0.5μmol) of the tetramethylated tetraamine disulfide (27) was introduced.The condenser was filled with acetone and dry ice, and the contents ofthe flask was cooled with a dry ice bath. Liquid ammonia was thenintroduced in the flask until about 25 mL was added. The solution wasstirred for several minutes and 36 mg (1.6 mmol) of sodium was added.The mixture was stirred for thirty minutes, and ammonium chloride (172mg) was cautiously added. The cooling bath was then removed to allow theammonia to evaporate. The white residue was dissolved with 45 mL ofMilli-Q water and concentrated hydrochloric acid was added to brougththe pH of this solution to 1. The mixture was extracted with ether (3×50mL) and the pH was raised to about 9 with 37% ammonium hydroxide. Thebasic solution was extracted four times with ether (50 mL). The organicphases were combined, washed successively with water (1×30 mL) and brine(2×30 mL). The ethereal phase was dried with anhydrous magnesium sulfateand filtered. The solvent was then removed under reduced pressure toyield 16 mg of a yellow oil. This oil was used as it is to perform the^(99m) technetium labeling study.

Note: The HPLC analysis of this oil has shown that it is a mixture oftwo component in a ratio of 2.5/1: the starting material and the reducedcompound. The HPLC conditions used for analysis were as followed: columnHamilton PRP-I 10 μm analytical, gradient of 10 to 50% of acetonitrile(with 0.1% TFA) in 40 minutes. Retention times: tetramethyl tetraaminedisulfide=7.73 min; tetramethyl tetraamine dithiol=21.55 min.

^(99m) Technetium Labeling of a Mixture Obtained From the Reduction ofN,N-dimethyl-2-(3,3,5,11,13,13-hexamethyl-1,2-dithia-5,8,11-triaza-cyclotridecan-8-yl)ethylamineExperiment A

In a 1.5 mL plastic conical vial 30 μL of a methanolic solution (˜2μg/μL) of the mixture obtained from the reduction ofN,N-dimethyl-2-(3,3,5,11,13,13-hexamethyl-1,2-dithia-5,8,11-triaza-cyclotridecan-8-yl)ethylamine,0.1 mL Tc-m99 tectnetium glucoheptonate (prepared by the reconstitutionof a vial from a FROSSTIMAGE GLUCOHEPTONATE kit with 1 mL of sodiumTc-m99 pertechnetate) were introduced. This solution was stirred for 20seconds with a vortex mixer and heated at 63° C. for 21/2 hours. Themixture was then analyzed by ITLC^(@) (Instant Thin LayerChromatography) ran in acetone and in normal saline and demonstratedthat 90% of the radioactivity was tagged with the chelate. HPLCanalysis§ of the labeling mixture showed the presence of threeradioactive products. The first radioactive compound was identified as^(99m) technetium glucoheptonate (R_(T) :2.59 min; 19%) while the twoothers are ^(99m) Tc-N₃ S₂ chelated species (R_(T) :20.72 and 23.08 min;68 and 8% respectively).

Experiment B

In a 1.5 mL plastic conical vial 30 μL of a methanolic solution (˜2μg/μL) of the mixture obtained from the reduction ofN,N-dimethyl-2-(3,3,5,11,13,13-hexamethyl-1,2-dithia-5,8,11-triaza-cyclotridecan-8-yl)ethylamine, 0.2 mL Tc-m99 technetium glucoheptonate (prepared by thereconstitution of a vial from a FROSSTIMAGE GLUCOHEPTONATE kit with 1 mLof sodium Tc-m99 pertechnetate) were introduced. This solution wasstirred for 20 seconds with a vortex mixer and heated at 65° C. for 1hour. The mixture was then analyzed by ITLC.sup.¶ (Instant Thin LayerChromatography) ran in acetone and in normal saline and demonstratedthat 85% of the radioactivity was tagged with the chelate. HPLCanalysis.sup.§ of the labeling mixture showed the presence of threeradioactive products. The first radioactive compound was identified asTc-m99 technetium glucoheptonate (R_(T) :2.72 min; 30%) while the twoothers are Tc-m99-N₃ S₂ chelated species (R_(T) :20.70 and 23.08 min; 56and 8% respectively).

.sup.¶ : Gelman SG chromatography paper.

.sup.§ : The high performance liquid chromatography analysis wasperformed with a Waters 625LC instrument equipped with a Hamilton PRP-I10 μm analytical column using a 10 to 50% acetonitrile (+TFA) gradientin 40 minutes and a flow rate of 1 mL/min.

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6. M. Apparu, S. Drouillard, J. P. Mathieu, A. DuMoulinet D'Hardemare,R. Pasqualini, and M. Vidal, AppI. Radial. Isot., 43 (5), 585-596(1992).

What is claimed is:
 1. A compound of the formula: ##STR4## wherein: R is selected from the group consisting of:hydrogen, loweralkyl of 1-4 carbon atoms, and loweralkyl carboxyl, wherein loweralkyl is 1-4 carbon atoms; R¹ is selected from the group consisting of:hydrogen, a sulfur protecting group, or where the two R¹ groups are replaced by a disulfide linkage; R² is selected from the group consisting of: ##STR5## n and m are independently an integer from 1-4; with the proviso that if R² is -N=C=S or -NH₂, and R is hydrogen or loweralkyl carboxyl, then the two R¹ groups are replaced by a disulfide linkage.
 2. The compound of claim 1 wherein the definition of R¹ the sulfur protecting group is selected from the group consisting of:p-loweralkyloxyl (1-4 carbon atoms) benzyl, and trityl.
 3. The compound of claim 2 wherein the definition of R1 the sulfur protecting group is p-methoxylbenzyl.
 4. The compound of claim 1 which is labeled with Tc-m99, Re-186 or Re-188.
 5. A compound of the formula: ##STR6## wherein: R is hydrogen;the two R¹ groups are replaced by a disulfide linkage; R² is: ##STR7## and n is
 2. 6. The compound of claim 5 which is labeled with Tc-m99, Re-186 or Re-188.
 7. A compound of the formula: ##STR8## wherein: R is CH₃ ;R¹ is p-methoxybenzyl; R² is: ##STR9## and n is
 1. 8. The compound of claim 7 which is labeled with Tc-m99, Re-186 or Re-188.
 9. A compound which is:4-isothiocyanato-N-[2-(3,3,5,11,13,13-hexamethyl-1,2-dithia-5,8,11-triazacyclotridecan-8-yl)ethyl]benzamide.
 10. The compound of claim 9 which is labeled with Tc-m99, Re-186 or Re-188. 